The present invention relates to a process for preparing cephalotaxane derivatives bearing a side chain.
The term xe2x80x9ccephalotaxanesxe2x80x9d refers to compounds or salts thereof which have a basic skeleton of formula 
where p is equal to 1 or 2 (it being possible for the two units to be identical or different and linked via a single bond or an oxygen atom), which can contain various oxygenated substituents (aliphatic or aromatic ethers, free or esterified alcohols, substituted or free enols and/or phenols, bridged ethers, and more generally any substituent usually encountered in the natural state on compounds of this type).
Harringtonines are alkaloids which are of high interest in anticancer chemotherapy, in particular on certain haematosarcomas which are multi-resistant to the existing therapies. The selectivity of harringtonines, which is based on a novel mechanism of action relating to protein synthesis, is such that this series is favoured with a great future in anticancer therapy.
Several literature compilations give a seemingly exhaustive review of all of the knowledge relating to cephalotaxanes, these compilations being, chronologically: [C. R. Smith, Jr, R. G. Powell and K. L. Mikolajczack, Cancer Treat. Rep., Vol. 60, 1157 (1976); C. R. Smith, Jr, L. Kenneth, K. L. Mikolajczack and R. G. Powell in xe2x80x9cAnticancer Agent Based on Natural Product Modelxe2x80x9d, 391 (1980); Liang Huang and Zhi Xue in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. XXIII (A. Brossi Ed.), 157 (1984); M. Suffness and G. A. Cordell in xe2x80x9cThe Alkaloids, Chemistry and Pharmacologyxe2x80x9d (A. Brossi Ed.), Vol. 25, 57-69, 295-298 (1 987); P. J. O""Dwyer, S. A. King, D. F. Hoth, M. Suffness and B. Leyland-Jones, Journal of Clinical Oncology, 1563 (1986); T. Hudlicky, L. D. Kwart and J. W. Reed, in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, 199 (1998)].
Antiparasitic activities, in particular on the haematozoon of malaria, have also been recognized [J. M. Whaun and N. D. Brown, Ann. Trop. Med. Par., Vol. 84, 229 (1990)].
Homo-harringtonine (HHT), the most active member of the series, is active at and above daily doses of 2.5 mg/m2 of body area per 24 hours, i.e., as a guide, at doses twenty times lower than that for Taxol(copyright). HHT has already undergone fourteen phase I and II clinical trials and it is the only known product capable of a 70% reinduction of full haematological remissions in patients suffering from chronic myeloid leukaemias that have become resistant to alpha-interferon [S. O""Brien, H. Kantarjian, M. Keating, M. Beran, C. Koler, L. E. Robertson, J. Hester, M. Rios, M. Andreeff and M. Talpaz, Blood, 332 (1995); Leukemia Insights, Vol. 3, No. 1 (1998)].
Harringtonines were extracted over 35 years ago from an exclusively Asiatic cephalotaxacea known as Cephalotaxus harringtonia, following the programme of research into novel anticancer agents in the plant kingdom developed by the National Cancer Institute. In fact, the Cephalotaxus alkaloids consist essentially (at least 50%) of cephalotaxine, a biosynthetic precursor of the harringtonines, the latter individually representing only a few per cent of the total alkaloids.
Besides their low concentration in the natural state in plant starting material, harringtonines are mixed with many congeners which have very similar chemical structures. Thus, in a high resolution high performance liquid chromatography (HPLC) chromatogram of a semi-purified alkaloid extract, no less than several tens of cephalotaxine esters are counted.
If we consider that:
on the one hand, harringtonines are generally relatively non-crystallogenic, as is suggested by the flexibility of their side chains, which are generally branched and aliphatic,
on the other hand, these esters, in particular harringtonine and homo-harringtonine, are contaminated with congeners which are themselves biologically active and very difficult to separate out, even by high resolution analytical HPLC,
the current state of the art does not allow these compounds to be produced viably on the industrial scale as regards the purity required for pharmaceutical active principles.
Although biosynthetically similar to the alkaloids of the genus Erythrina, cephalotaxanes are alkaloids which have a unique structure in nature, encountered only in the genus Cephalotaxus, which is the only genus of the Cephalotaxacea family. On the other hand, the side chains of the various harringtonine congeners are all derived from the methyl hemiester of the primary carboxyl of (2R) citramalic acid 3a (see Scheme 1 attached) by substitution of the tertiary methyl using alkyl or aralkyl radicals which may themselves be unsubstituted or substituted with tertiary hydroxyls, it then being possible for the latter to form a cyclic ether with a tertiary alcohol (anhydro derivatives).
The attached Scheme 1 shows the main examples of harringtonine congeners, which all have significant cytostatic activity to different degrees. None of the artificial analogous cephalotaxine esters synthesized hitherto in the literature has at least the sub-structure 3b (see Scheme 1) and lack significant cytostatic activity.
It is worthwhile pointing out that, although botanically very similar to the Cephalotaxaceas, Taxaceas contain triterpene alkaloids (taxines), accompanied by non-alkaloid triterpenes, taxanes, which are also of unique structure in nature. Although they are completely different from taxanes in terms of chemical structures and anticancer mechanism of activity, the harringtonines have analogy with taxanes in more than one respect:
they have cytostatic properties,
they consist of a polycyclic skeleton, an inactive biosynthetic precursor of the complete structure, onto which is grafted a side chain containing a similar combination of hydrophilic and hydrophobic substituents,
the polycyclic part of the taxanes (baccatins in the broad sense) and of the harringtonines (cephalotaxines) is relatively abundant in renewable parts of the plant, whereas the active molecules (harringtonines and taxanes) are ten to one hundred times less abundant therein,
the plum yew (Cephalotaxus) is a rare tree, even rarer than the yew (Taxus), and is much less ubiquitous than the latter.
It results from the above facts that, following the manner of the semi-synthesis of taxanes by adding a synthetic chain to a 10-deacetylbaccatin III of extracted origin, the asymmetric semi-synthesis of harringtonines by esterification of a cephalotaxine of natural origin is of considerable medical and economic value. Furthermore, the current population of Cephalotaxus is relatively reduced even in their original habitat. Thus, during its importation into Europe for ornamental purposes last century, Cephalotaxus harringtonia was already no longer present in spontaneous form in eastern China and in northern Japan. The use of a precursor present in a renewable part of the tree (the leaf) in order to prepare homo-harringtonine semi-synthetically is thus of considerable environmental interest, all the more so since the total synthesis of optically active cephalotaxine has not been achieved hitherto, despite the extensive synthetic studies carried out in this respect (a certain number of laborious syntheses of racemic cephalotaxine containing 10 to 15 steps have, however, been carried out: see bibliographic review above).
Consider that several hundred tonnes per year of this rare and very slow-growing tree (even slower growing than Taxus sp.) need to be extracted to satisfy the current market needs for homo-harringtonine (several kilograms per year), whereas the semi-synthesis would consume only a few tonnes of renewable parts of the tree (leaves). Furthermore, homo-harringtonine (HHT) of natural origin currently available on the active principles market is contaminated with its congeners, which, on account of their structural similarity, are very difficult to separate, even by xe2x80x9cpreparativexe2x80x9d high performance liquid chromatography.
First of all, it should be noted that since the use of cephalotaxine itself as a source for semi-synthesis has not yet been economically justified, no process for selectively extracting this substance has been described hitherto. Moreover, among the active compounds, only harringtonine and isoharringtonine have been the subject of American patent applications for their preparation by extraction [R. G. Powell et al., U.S. Pat. No. 3,793,454 and U.S. Pat. No. 3,870,727]. Harringtonine has been the subject of a Japanese patent [JP 58-032,880] and deoxyharringtonine has been the subject of an American patent [U.S. Pat. No. 3,959,312]. As regards the preparation of homo-harringtonine itself, it has been the subject of only a few semi-synthetic studies [T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, 199 (1998)], but no patent application has been made regarding a semi-synthesis process or even an extraction process.
Another aspect which gives the present invention an even greater advantage is that cephalotaxine can serve as a springboard for the synthesis of cephalotaxoids and harringtoids which are useful for antitumour (cancerous and non-cancerous tumours), antiparasitic, antifungal, antiviral and antibacterial chemotherapies. Harringtonines consist of a complex alkaloid polycyclic alcohol (cephalotaxine), esterified with a side chain, in isolation having no more biological activity than cephalotaxine, but essential for the biological activity of the whole. Saponification of the side chain under harsh conditions leads to the cephalotaxine free base and to harringtonic acids. The attachment of the side chains takes place at the end of the biosynthesis. It has been demonstrated that catabolism leading to this reaction could be triggered in vivo under the influence of environmental or physiological stress exerted on the plant [N. E. Delfel, Phytochemistry, 403 (1980)].
Cephalotaxine, the polycyclic part consisting of 5 fused rings, has a novel arrangement which is unique in nature, i.e. a benzodioxoazepine onto which is fused a spiropyrrolidinopentenediol system. Cephalotaxane contains four asymmetric centres: three xe2x80x9casymmetric carbonsxe2x80x9d and a heterocyclic tertiary aminic nitrogen. The only reactive function is a secondary alcohol located in position 3, the methyl enol ether located in position 2 being potentially sensitive to proton attack. The whole forms a pseudohelical structure encaging the hydroxyl in the tube formed by the tetrahydrazepine. The base cephalotaxine readily forms highly crystallogenic stable salts (for example hydrochlorides and perhydrochlorides).
This alkaloid is relatively insensitive to basic media. On the other hand, several authors describe a certain level of sensitivity to acids and to quaternization of the nitrogen with methyl iodide, leading to a racemization by simultaneous inversion of the 3 asymmetric centres and of the nitrogen [D. J. Abraham, R. D. Rosensten and E. L. McGandy, Tetrahedron Letters, 4085 (1969)]. However, a period of several days in solution at pH 1-4 at 20xc2x0 C. leaves this structure intact (personal observation).
This compound and its congeners which are not O-acylated in position 3 are biologically inactive.
All the side chains for harringtonines which have significant biological activity contain in common the 2-alkyl-2-carbomethoxymethyl-2-hydroxyacetyl unit. The alkyl chain, of variable length, has at the end either branching constituting an isopropyl bearing (harringtonine HT and homo-harringtonine HHT) or not bearing (deoxy-homo-harringtonine DHT) a tertiary alcohol, or a phenyl radical (for example the neoharringtonine series most recently isolated). In the case of the anhydroharringtonines, the chain can be closed by dehydration between its two tertiary alcohols, for example forming a substituted tetrahydropyran ring. The tertiary carboxyl of this complex diester is borne by the single hydroxyl of the cephalotaxine. The only chiral centre on the side chain is located (to the ester junction. It contains, besides the first secondary chain, a hydroxyl which, on account of its tertiary nature, does not have the possibility of epimerizing.
Scheme 2 attached shows synthetically the known processes for preparing harringtonines.
Several semi-syntheses of natural cephalotaxine esters and several series of analogues, which have simplified chains but give these analogues reduced cytotoxic activity, have been described hitherto, in particular those of deoxyharringtonine and of isoharringtonine. Most of them relate to simpler and less functionalized esters than those constituting HT and HHT, the esters which are most useful in chemotherapy [for example, deoxyharringtonine, isoharringtonine, T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987)].
All the literature from 1972 to the present date [Mikolajczack et al., Tetrahedron, 1995 (1972); T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxane 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3e totally preformed to give a harringtonine 4b, i.e. the conversion 2a+3e (4b as described in the example featured in the scheme below 
Most of the syntheses described hitherto thus involve binding of the secondary side chain xe2x80x94CH2CO2Me, i.e.:
1st) by the Reformatsky reaction between methyl bromoacetate and the carbonyl (real or potential) on the side chain prebound to cephalotaxine, in the presence of zinc, or
2nd) by prior formation of an organolithium reagent.
All the syntheses described thus consist in esterifying cephalotaxine using the (-keto alkanoyl chloride 7 lacking the end hydroxyl and containing neither the secondary chain located (to the tertiary carboxyl, nor the tertiary hydroxyl (to the carboxyl, to give 8 which is then converted into a harringtonine 4a, according to the reaction described below. 
In formula 8, CTXxe2x80x94 represents the cephalotaxyl radical of formula: 
It should be noted that this (-hydroxyalkylation, which at the same time creates the chiral centre on the side chain, has never been achieved asymimetrically. A few synthetic routes involve an esterification of cephalotaxine with a substituted hemisuccinyl chloride, optionally followed by subsequent introduction of the tertiary hydroxyl(s).
No O-acylation of cephalotaxine, using totally preformed and functionalized chiral chain precursors (to the tertiary carboxyl, has thus been achi eved hitherto [T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, pages 661 to 675 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, pages 224 to 236 (1998)].
Consequently, the methods for preparing harringtonines by semi-synthesis, which have been described to date in the existing art, have the following drawbacks:
absence of stereoselectivity,
poor convergence,
mediocre yields,
functionalization and construction of the chain on a rare and expensive substrate,
chiral homo-harringtonine not obtained to date.
Since cephalotaxine is present in nature in partially racemized form [personal observation; Huang et al., Scientia Sinica, Vol. XXIII, 835 (1980)], the processes of the prior art which use a natural cephalotaxine as starting material can only theoretically result in partially racemized harringtonines. The present invention thus has the advantage of obtaining enantiomerically pure harringtonines even from racemic cephalotaxine, since:
1st) the asymmetric centre on the side chain is created prior to the esterification step, i.e. the side chain precursor can be obtained in enantiomerically pure form prior to being attached,
2nd) the diastereoisomers obtained in the case of a racemic cephalotaxine can be separated by chromatography.
The present invention consists in:
esterifying the hindered free alcohol of a cephalotaxine or alternatively the corresponding metal alkoxide, using a chain in the form of a suitably substituted tertiary carboxylic oxacycloalkane acid which is totally preformed both in terms of the skeleton and in terms of the functionalization, in order to prepare anhydro-homo-harringtonic acids by semi-synthesis.
opening the cyclic side chains thus formed in order to obtain the corresponding diols, i.e. the harringtonines (defined above).
describing a new preparation for all of the diastereoisomers of the dihydroxylated side chains of the harringtonines in a dehydrated cyclic form (anhydroharringtonic acids) or in which the two hydroxyl groups are protected together by difunctional protecting groups forming a ring.
resolving all of the harringtonic and anhydroharringtonic acids, in order to couple them separately with the cephalotaxines.
One part of the present invention thus consists in synthesizing, in particular, anhydroharringtonine, harringtonine, anhydro-homo-harringtonine and homo-harringtonine.
The present invention also relates to esterifying cephalotaxines or metal alkoxides thereof with N-alkyl- and N-carbamoyl-2-alkylisoserine.